Drug target for preventing and treating periodontal disease, improving healing of periodontal wounds and promoting oral health

ABSTRACT

The present invention relates to the use of a component of the plasminogen-activating pathway and use of compounds which have the capacity to activate plasminogen directly or via the plasminogen-activating pathway, for prophylaxis, prevention and treatment of periodontal disease including peri-implantitis, healing of periodontal wounds and prompting oral health in human and non-human subjects.

FIELD OF INVENTION

This invention relates to compound and methods for prophylaxis,prevention and/or treatment of infectious periodontal disease e.g.gingivitis and periodontitis, and necrotic conditions affecting the gumtissue, relates to promoting oral health in general, and also relates toimproving healing of periodontal wounds such as surgical wounds locally.In particular, the invention relates to a novel method of preventing andtreating infectious periodontal disease, promoting oral health andimproving healing of periodontal wounds.

BACKGROUND

Periodontal Disease

Periodontal disease is a chronic inflammatory disease that affects thetissues that support and anchor the teeth, also known as theperiodontium. It is caused by the imbalanced interplay between thespecific subgingival microorganisms and the host immune and inflammatoryresponse (1). It affects nearly three-quarters of the adult populationsand is regarded as one of the most common diseases to human being. Thetissues that are involved in periodontal diseases are the gums, whichinclude the gingiva, the periodontal ligament, the cementum, and thealveolar bone (FIG. 1). The gingiva is a pink-colored keratinized mucusmembrane that covers parts of the teeth and part of the alveolar bone.The periodontal ligament is the main part of the gums. The cementum is acalcified structure that covers the lower parts of the teeth. Thealveolar bone is a set of ridges from the jaw bones (maxillary andmandible) in which the teeth are embedded. The area where periodontaldisease is initiated is the gingival sulcus, a pocket between the teethand the gums.

Infection, inflammation and subsequent host defense and wound healingare all hallmarks of periodontal disease. This disease begins as a mixedbacterial infection in the gingiva surrounding teeth (2). In the healthymouth, more than 500 species of microorganisms have been found. Inperiodontal diseases, several potential periodontal pathogens have beenstudied including Porphyromonas gingivalis, Campylobacter rectus,Actinobacillus actinomicetemcomitans, and Fusobacterium nucleatum, whichare considered to represent a significant portion of the pathogenicmicrobiota. These microorganisms can induce several factors, such asIL-1, IL-6, TNF, as well as enzymes, in host cells which directly orindirectly are thought to cause irreversible tissue destructionincluding the destruction of the gums, the alveolar bone, the outerlayer of the tooth root and eventually leads to tooth loss. Furthermore,serious periodontal disease can lead to bad breath, heart disease andstroke, diabetes, respiratory diseases and premature delivery duringpregnancy. There are other pathogenic factors such as smoking/tobaccouse, genetics, pregnancy and puberty, stress, medications, diabetes,poor nutrition and other systemic diseases.

Another form of infectious destruction of the alveolar bone, closelyresembling periodontitis, namely Periimplantitis, can occur aftersurgical implantation of an alloplastic material into the jaws. Theimplantation method is often referred to as osseointegration (3), whichentails close contact between the alloplastic material, i.e. the dentalimplant (often made of titanium), and the living bone. The method isused to restore occlusion subsequent to the loss of natural teeth and isnow a standard method for treating edentulism. A principle differencebetween the osseointegrated dental implant and the natural tooth is theabsence of a true periodontium around the implant. While the normaltooth is suspended in a meshwork of collagenous fibers that allows for aphysiological mobility of the tooth within the alveolar bone, the dentalimplant is firmly connected to the bone without intervening soft tissue.Despite this major dissimilarity in attachment to the bone tissue, thepathological changes at teeth and implants during infection share manykey features such as infection via biofilm formation and colonization,inflammatory response, as well as immunological defence. Thus,periimplantitis is an inflammatory/infectious process affecting thetissues around an osseointegrated implant in function, resulting in lossof supporting bone. Periimplantitis may lead to complete disintegrationand implant loss even if extensive treatment aiming at resolving theperiimplant infection has been performed. Periimplantitis also happensas reversible inflammatory/infectious changes of the peri-implant softtissues without any bone loss. The prevalence of periimplantitis in thesoft tissue has been reported in the range of 8-44%, while frequency ofperiimplantitis in the bone has been reported in the range of 1-19%. Thewide ranges for the frequencies seem to be due to differences indefining the entity, at least in part. The frequency of periimplantitisis most likely related to the number of years implants have been worn.Since dental implant treatment was introduced comparatively recently,the numbers will probably increase over the years. Considering the largesimilarities in the inflammatory response and the immunological defenceagainst infection at teeth and dental implants periimplantitis could beregarded as a form of periodontal disease affecting implantedalloplastic material.

Periodontal disease is an important aspect of general oral health. Oralhealth refers to the status of health of the oral and related tissueswhich enables an individual to eat, speak and socialize without activedisease, discomfort or embarrassment and which contributes to generalwell-being. Major indications of oral health include the bacterial florain the saliva and gum tissue, as well as the tissue necrosis andinflammation in the gum tissue. Oral health is integral to generalhealth and should not be considered in isolation.

Antibiotics and other antimicrobial drugs have been widely used intreatment of infectious diseases since the World War II era. The successof antimicrobials against disease-causing microbes is among modernmedicine's great achievements. However, many antimicrobials are not aseffective as they used to be. A key factor in the development ofantibiotic resistance is the ability of infectious organisms to adaptquickly to new environmental conditions. Over time, some bacteria havedeveloped ways to circumvent the effects of antibiotics. Widespread useof antibiotics is thought to have spurred evolutionarily adaptationsthat enable bacteria to survive these once so powerful drugs.Ultimately, the increasing difficulty in fighting off microbes leads toan increased risk of acquiring infections in a hospital or othersetting. Drug resistance is an especially difficult problem forhospitals harboring critically ill patients who are less able to fightoff infections without the help of antibiotics. Therefore, there is anincreasing awareness that novel therapeutic strategies are highly neededto improve the infection defense against infection.

Treatment of periodontal disease includes conservative (non-surgical)methods and surgical methods. Conservative treatment consists of deepcleanings known as scaling and rootplaning as well as gingivalcurettage. This treatment is aimed to remove the biofilm colonizing theaffected root surfaces and reestablish an environment where healing canoccur. Accompanied with good oral hygiene this will maintain healthynormal gums. Surgical periodontal treatment consists of osseous (bone)surgery, gingival/periodontal grafts, gingival flap procedure,frenectomy, gingivectomy, guided tissue regeneration/bone augmentation.However, despite the various therapeutic methods that have successfullyimproved the treatment of periodontal disease, great challenges in oralhealth still exist. Such challenging factors include the increasingresistance of oral bacteria against antibiotics, the needs for simplermethods to improve oral health in general, the expensive and tediousdental care procedure, the stressful modern life and the heavier dentalburden in under-privileged groups in developed and developing countries.Therefore novel methods of preventing and treating periodontal disease,promoting oral health and improving healing of periodontal wounds are ingreat needs.

Necrosis

Necrosis is the name given to unprogrammed or accidental death of cellsand living tissue. It is less orderly than apoptosis, which are part ofprogrammed cell death. In contrast with apoptosis, cleanup of celldebris by phagocytes of the immune system is generally more difficult,as the disorderly cell death generally does not send “eat-me” cellsignals which tell nearby phagocytes to engulf the dying cell. This lackof signalling makes it harder for the immune system to locate andrecycle dead cells which have died through necrosis than if the cell hadundergone apoptosis. The release of intracellular content after cellularmembrane damage is cause of inflammation in necrosis. There are manycauses of necrosis including injury, infection, cancer, infarction,invenomation and inflammation. Severe damage to one essential system inthe cell leads to secondary damage to other systems, a so-called“cascade of effects”. Necrosis is caused by special enzymes that arereleased by lysosomes which are capable of digesting cell components orthe entire cell itself. The injuries received by the cell may compromisethe lysosome membrane, or may set off an unorganized chain reactionwhich causes the release in enzymes. Unlike in apoptosis, cells that dieby necrosis may release harmful chemicals that damage other cells.Necrosis of biopsy material is halted by fixation or freezing.

Necrosis occurs in certain types of periodontal disease. Necrotizinggingivitis is an inflammatory destructive gingival conditioncharacterized by interproximal necrotic ulcers, spontaneous bleeding,rapid onset of pain and bad odor. Unless properly treated, necrotizinggingivitis has a marked tendency for recurrence and lead to considerableloss of periodontal support.

Currently there are four major therapeutic methods to cure necrosis. Thefirst is surgical., which is the most rapid, and therefore isrecommended when large necrotic areas or thick scars are present. Thesecond is mechanical, which includes hydrotherapy, dextranomers andwound irrigation. The third is enzymatical, the enzyme used is mainlycollagenase (eg: Santyl), however, the effect is too slow when infectionpresents; and the fourth is through autolytic method, which is viaenzymes in wound fluid but the effect is extremely slow. However, noneof the four treatment methods could give a functional and aestheticallysatisfactory necrosis removal and tissue remodeling. Therefore, a noveltherapeutic strategy is in great need in order to achieve a successfulremoval of necrosis.

The Plasminogen-Activation System

Plasmin is the key component of the PA system. It is a broad-spectrumprotease which has the ability to degrade several components of the ECMincluding fibrin, gelatin, fibronectin, laminin and proteoglycans (4).In addition, plasmin can convert some pro-matrix metalloproteinases(pro-MMPs) to active MMPs. It has therefore been suggested that plasminmay be an important upstream regulator of extracellular proteolysis(5;6). Plasmin is formed from the zymogen plasminogen throughproteolytic cleavage by either of two physiological PAs, tPA or uPA. Asplasminogen is present in plasma and other body fluids at relativelyhigh levels, the regulation of the PA system occurs mainly at the levelof synthesis and activity of the PAs. Synthesis of the components of thePA system is highly regulated by different factors such as hormones,growth factors and cytokines. In addition, there exist specificphysiological inhibitors of plasmin and PAs. The main inhibitor ofplasmin is α₂-antiplasmin. The activity of PAs is regulated by PAI-1,which inhibits both uPA and tPA, and PAI-2, which inhibits mainly uPA.Certain cells also have a specific cell-surface receptor for uPA (uPAR)that can direct proteolytic activity to the cell surface (8;9).

Plasminogen is a single-chain glycoprotein consisting of 790 amino acidswith a molecular mass of approximately 92 kDa (7;8). Plasminogen ismainly synthesized in the liver and is abundant in most extracellularfluids. In plasma the concentration of plasminogen is approximately 2μM. Plasminogen therefore constitutes a large potential source ofproteolytic activity in tissues and body fluids (9;10). Plasminogenexists in two molecular forms: Glu-plasminogen and Lys-plasminogen. Thenative secreted and uncleaved form has an amino-terminal (N-terminal)glutamic acid and is therefore designated Glu-plasminogen. However, inthe presence of plasmin, Glu-plasminogen is cleaved at Lys⁷⁶-Lys⁷⁷ tobecome Lys-plasminogen. Compared to Glu-plasminogen, Lys-plasminogen hasa higher affinity for fibrin and is activated by PAs at a higher rate.These two forms of plasminogen can be cleaved at the Arg⁵⁶⁰-Val⁵⁶¹peptide bond by uPA or tPA, resulting in the formation of thedisulphide-linked two-chain protease plasmin (11). The amino-terminalpart of plasminogen contains five homologous triple-loops, so-calledkringles, and the carboxyl-terminal part contains the protease domain.Some of the kringles contain lysine-binding sites which mediate thespecific interaction of plasminogen with fibrin and its inhibitor α₂-AP.A novel and interesting finding is that a 38-kDa fragment ofplasminogen, consisting of kringles 1-4, is a potent inhibitor ofangiogenesis. This fragment is termed angiostatin and can be generatedfrom plasminogen through proteolytic cleavage by several MMPs.

The main substrate for plasmin is fibrin, and dissolution of fibrin ispivotal for prevention of pathological blood clot formation (12).Plasmin also has substrate specificities for several other components ofthe ECM, including laminin, fibronectin, proteoglycans and gelatin,indicating that plasmin also plays an important role in ECM remodeling(8;13;14). Indirectly, plasmin can also degrade additional components ofthe ECM through its ability to convert some pro-MMPs to active MMPs,including MMP-1, MMP-2, MMP-3 and MMP-9. It has therefore been suggestedthat plasmin may be an important upstream regulator of extracellularproteolysis (15). In addition, plasmin has the ability to activatelatent forms of certain growth factors (16-18). In vitro, plasmin alsocleaves components of the complement system and thereby releasechemotactic complement fragments.

The PA system has been suggested to be involved at several stages and byvarious mechanisms during bacterial invasion (19). A vast number ofpathogens express plasmin(ogen) receptors (20;21). Bacteria alsoinfluence the secretion of PAs and their inhibitors from mammalian cells(22;23). For instance, production of uPA has been found to be enhancedin cells infected by various bacteria (24). To date, in vivo evidencefor a role of plasminogen activation in pathogenesis exists in a fewbacteria such as Yersinia pestis, Borrelia, and group A streptococci.

Binding of plasminogen to receptors present on the surfaces of somebacteria convert these bacteria into proteolytic organisms. InGram-negative bacteria, the filamentous surface appendages form a majorgroup of plasminogen receptors (25;26). In Gram-positive bacteria,surface-bound molecules have been identified as plasminogen receptors(27;28). As a consequence, plasmin can be generated on the surface ofmicroorganisms such as Haemophilus influenzae, Salmonella typhimurium,Streptococcus pneumoniae, Yersinia pestis, and Borrelia burgdorferi,which can lead to a degradation of mammalian ECM. Furthermore, bacterialproteases may also directly activate latent pro-collagenases orinactivate protease inhibitors in human plasma, and thus contribute totissue damage and bacterial spread across tissue barriers (29;30).

Models of Periodontal Disease and Periodontal Wounds

Models of periodontal disease include spontaneous type and induced type.The periodontal tissue is exposed to a microbe-rich environment.Bacterial invasion and subsequent host defense in the oral cavity occursconstantly and normally remain in balance. Disruption of thishost-bacterial balance causes various types of periodontal disease. Thiscould be due to an imbalance between the oral microbiota, alterations inphagocyte function and/or specific immune response. Severe periodontaldisease occurs in approximately 2% of US adolescents and inapproximately 20% of US adults.

Inducing periodontal disease by certain bacterial species providesdefined models for periodontitis. Commonly used periodontal pathogensinclude Porphyromonas gingivalis, Campylobacter rectus, Actinobacillusactinomycetemcomitans, and Fusobacterium nucleatum, which are consideredto represent a significant portion of the pathogenic microbiota. Theypossess or can induce in host cells several factors. such as IL-1, WL-6,tumor necrosis factor, surface-associated proteins, fimbriae, vesicles,toxins, and enzymes, which are thought to cause, directly or indirectly,irreversible loss of periodontal supportive tissues.

Periodontal wounds are commonly seen, especially during periodontalsurgery. Periodontal wound model can be established by inducingincisional wounds at the gum tissue in the mice. Thereafter the healingpattern of the wounds and the effects of the candidate drug or compoundscan be evaluated.

Current method for treating infections such as necrosis as well asperiodontal disease have drawbacks as discussed above. Thus, there isstill a need in the art for improved strategies and means for treatingperiodontal disease and improving oral health.

SUMMARY OF THE INVENTION

The present invention relates to the novel discovery that components ofthe plasminogen-activation pathway, and compounds with the capacity toactivate plasminogen can be used for new and improved strategies forpreventing and treating periodontal disease and tissue necrosis, forhealing of periodontal wounds (such as surgical wounds) and forpromoting oral health in general. The administration of plasminogenand/or other members of the plasminogen-activation pathway or compoundswith the capacity to activate plasminogen play a pluripotent role inprotecting against bacterial-induced infection and promoting healing ofperiodontal wounds by activating inflammatory cells, acceleratingmigration of keratinocytes, killing bacteria, removing necrotic tissueand enhancing cytokine expression. The extensive occurrence ofperiodontal disease in plasminogen-deficient mice under naturalconditions also provides an excellent animal model to for studyingperiodontal disease, and screening methods for identifying andevaluating new drugs and treatment methods for various aspects ofperiodontal disease, periodontal wound improvement and prompt oralhealth in general.

Accordingly, the present invention provides the use of an active agentor compound that is a component of the plasminogen-activating pathway orhas the capacity to activate plasminogen directly or via theplasminogen-activating pathway for the manufacture of a pharmaceuticalcomposition comprising an effective amount of the compound/agent, or acombination of two or more such agents/compounds, for the prophylaxis,prevention and/or treatment of periodontal disease especially infectiousperiodontal disease, and/or removal of necrosis in the gum tissue, in asubject in need of such treatment, Preferably, the active agent isselected from plasminogen activators, tPA, uPA, streptokinase,saruplase, alteplase, reteplase, tenecteplase, anistreplase, monteplase,lanoteplase, pamiteplase, staphylokinase and recombinant forms andvariants of the components of the plasminogen-activating pathway. Morepreferably, the active agent is selected from plasmin or plasminogen andtheir derivatives e.g. kringle domains of plasmin or plasminogen,protein fragments of plasmin or plasminogen, mini-plasminogen andmini-plasmin as well as the synthetic derivatives of plasmin orplasminogen. Most preferably, the active agent is plasminogen and itsderivatives. The active agent can be administered by any route ofadministration known in the art. Preferred, non-limiting, routes ofadministration include topical application, intra-gingival injection andintravenous injection. The agent may also be present in a wound dressingapplied onto the infected area of periodontal tissue, if possible, fromwhich it is transferred to the infected site of periodontal tissue. Thecomposition may be part of a gel, lotion, balm, paste (toothpaste),gargling solution (mouthwash solution) or wound dressing

The present invention also provides the use of a compound or activeagent that is a component of the plasminogen-activating pathway or acompound which has the capacity to activate plasminogen directly or viathe plasminogen-activating pathway for the manufacture of apharmaceutical composition comprising an effective amount of suchcompound/agent, or a combination of tow or more such compounds/agents.for improving resolution and/or promoting the healing of periodontalwounds and surgical periodontal wounds, especially infectiousperiodontal wounds and surgical infectious periodontal wounds in asubject in need of such treatment. Preferably, the active agent isselected from plasminogen activators, tPA, uPA, streptokinase,saruplase, alteplase, reteplase, tenecteplase, anistreplase, monteplase,lanoteplase, pamiteplase, staphylokinase and recombinant forms andvariants of the components of the plasminogen-activating pathway. Morepreferably, the active agent is selected from plasmin or plasminogen.Most preferably, the active agent is plasminogen. The active agent canbe administered by any route of administration known in the art.Preferred, non-limiting, routes of administration include topicalapplication, intra-gingival injection and intravenous injection. Theagent may also be present in a wound dressing, a gel, lotion, balm,paste, mouthwash solution and toothpaste applied onto the wounded areaof periodontal tissue. if possible, from which it is transferred to thewounded site of periodontal tissue.

Furthermore, the present invention provides a method of prompting oralhealth, comprising administering a composition comprising an activeagent which is a component of the plasminogen-activation pathway or acompound with the capacity to activate plasminogen, or a combination oftwo or more such agents. Preferably, the active agent is selected fromplasminogen activators, tPA, uPA, streptokinase, saruplase, alteplase,reteplase, tenecteplase, anistreplase, monteplase, lanoteplase,pamiteplase, staphylokinase and recombinant forms and variants of thecomponents of the plasminogen-activating pathway. More preferably, theactive agent is selected from plasmin or plasminogen. Most preferably,the active agent is plasminogen, such as Glu-plasminogen orLys-plasminogen. The active agent can be administered by any route ofadministration known in the art. The composition may be part of a gel,lotion, balm, paste, or dressing. Preferred, non-limiting, routes ofadministration include topical application such as tooth paste or theusage of gargling solution (mouthwash solution) which can be used forstrengthening teeth against decay and prompting oral health.

The invention also provides for a method of initiating the host defensefor treating periodontal disease, especially infectious periodontaldisease, in conditions where host defense is retarded or impaired,comprising administering an active ingredient which is plasmin orplasminogen. In a particular embodiment, the method of the invention canbe used for improving host defense against periodontal disease inconditions of local or systemic deficiency/impairment of plasmin orplasminogen.

In another embodiment, the invention provides a method for prophylaxis,prevention and treatment of periodontal disease, especially infectiousperiodontal disease, improving healing of periodontal wounds such assurgical wounds and promoting oral health in human or non-human subjectsby administering a compound or drug which is plasminogen or plasmin andtheir derivatives, an activator of plasminogen, or a compound enhancingthe activity of plasmin. Preferably, the compound is administeredlocally to attain a high concentration in the infected area.

Moreover, the invention provides for a method for reducing or preventingoral necrosis formation by administering a composition comprising localor systemic administration of a composition comprising a compound whichis a component of the plasminogen activation pathway or compounds withthe capacity to activate plasminogen. The composition may be part of agel, lotion, balm, paste, or wound dressing. Alternatively, thecomposition may be administered systemically. In one embodiment, themethod of the invention is applied in conjunction with plastic surgeryin the periodontal tissue to reduce the occurrence and the formation ofinfection, ulcer and necrosis.

In another embodiment, the invention provides a pharmaceuticalcomposition for the treatment, prophylaxis and prevention of periodontaldisease, especially infectious periodontal disease, comprising aneffective amount of a compound which is a component of theplasminogen-activating pathway or compounds with the capacity toactivate plasminogen. The component of the plasminogen-activatingpathway can be selected from plasminogen, Lys-plasminogen,Glu-plasminogen, plasmin, kringle domains of plasminogen and plasmin,mini-plasminogen, mini-plasmin, plasminogen activators, tPA, and uPA.Preferably the component of the plasminogen-activating pathway isplasminogen or plasmin. The compounds with the capacity to activateplasminogen can be selected from streptokinase, saruplase, alteplase,reteplase, tenecteplase, anistreplase, monteplase, lanoteplase,pamiteplase, staphylokinase and recombinant forms and variants of thecomponents of the plasminogen-activating pathway.

In a further embodiment, the invention provides a method forprophylaxis, prevention and/or treatment of periodontal disease,especially infectious periodontal disease, comprising administering apharmaceutical composition comprising an effective amount of a compoundaccording to claims 1-16, which compound is a component of theplasminogen-activating pathway or which has the capacity to activateplasminogen directly or via the plasminogen-activating pathway to asubject in need of such treatment. The component of theplasminogen-activating pathway can be selected from plasminogen,Lys-plasminogen, Glu-plasminogen, plasmin, kringle domains ofplasminogen and plasmin, mini-plasminogen, mini-plasmin, plasminogenactivators, tPA, and uPA. Preferably the component of theplasminogen-activating pathway is plasminogen or plasmin. The compoundwith the capacity to activate plasminogen can be selected fromstreptokinase, saruplase, alteplase, reteplase, tenecteplase,anistreplase, monteplase, lanoteplase, pamiteplase, staphylokinase andrecombinant forms and variants of the components of theplasminogen-activating pathway.

In yet another embodiment, the invention provides a pharmaceuticalcomposition for promoting the healing of periodontal wounds, especiallyinfectious periodontal wounds, which comprises an effective amount of acomponent of the plasminogen-activating pathway or a compound with thecapacity to activate plasminogen. The component of theplasminogen-activating pathway can be selected from plasminogen,Lys-plasminogen, Glu-plasminogen, plasmin, kringle domains ofplasminogen and plasmin, mini-plasminogen, mini-plasmin, plasminogenactivators, tPA, and uPA. Preferably the component of theplasminogen-activating pathway is plasminogen or plasmin. The compoundwith the capacity to activate plasminogen can be selected fromstreptokinase, saruplase, alteplase, reteplase, tenecteplase,anistreplase, monteplase, lanoteplase, pamiteplase, staphylokinase andrecombinant forms and variants of the components of theplasminogen-activating pathway.

In a further embodiment, the invention provides a method for promotingthe healing of periodontal wounds, especially infectious periodontalwounds, comprising administering a pharmaceutical composition comprisingan effective amount of compound, which is a component of theplasminogen-activating pathway or which has the capacity to activateplasminogen directly or via the plasminogen-activating pathway to asubject in need of such treatment. The component of theplasminogen-activating pathway can be selected from plasminogen,Lys-plasminogen, Glu-plasminogen, plasmin, kringle domains ofplasminogen and plasmin, mini-plasminogen, mini-plasmin, plasminogenactivators, tPA, and uPA. Preferably the component of theplasminogen-activating pathway is plasminogen or plasmin. The compoundwith the capacity to activate plasminogen can be selected fromstreptokinase, saruplase, alteplase, reteplase, tenecteplase,anistreplase, monteplase, lanoteplase, pamiteplase, staphylokinase andrecombinant forms and variants of the components of theplasminogen-activating pathway.

DETAILED DESCRIPTION OF THE INVENTION

Improvement of Preventing and Treating Periodontal Disease, HealingPeriodontal Wounds and Maintaining Oral Health

According to the invention, providing or enhancing the levels ofplasminogen and/or plasmin can be used for prophylaxis, prevention andtreatment of periodontal disease, accelerating the healing ofperiodontal wounds and prompting the oral health. This may beaccomplished in many different ways. For instance, by treating a patientwith active agents, drugs, hormones, cytokines, antibodies, or othercompounds that up-regulate the expression of plasmin, plasminogen, orplasminogen-activators; reduce the degradation of either of thesecomponents; the local or systemic levels of plasminogen and/or plasmincan be increased. In another embodiment, local plasmin or plasminogenlevel is increased by directly applying plasmin/plasminogen proteins andtheir derivatives. In yet another embodiment, plasmin activity isenhanced by administration of an activator of plasmin or plasminogen. Infurther another embodiment, an artificial, a recombinant or a bacterialplasminogen activator such as streptokinase and staphylokinase is used.In further another embodiment, a fragment of plasminogen proteinsequence such as synthetic peptides, kringle domains miniplasminogen orminiplasmin is used.

Components of the plasminogen-activation pathway or compounds with thecapacity to activate plasminogen may be produced by purifying thecomponent(s) or compounds from bacteria, humans, or other animals, or byrecombinant production in yeast such as S. cerevisiae, in bacteria sucha E. coli, and in mammalian cell line such as Chinese Hamster Ovary cellline. The component may be wild-type or modified/mutated. Fragments ofthe component which retain at least a part of the desired activity ofthe full-length component may also be used. In a preferred embodiment, asubstantially pure preparation of human plasminogen is used. In anotherpreferred embodiment, a substantially pure preparation of human plasminis used. In further another preferred embodiment, a substantially purepreparation of miniplasminogen, miniplasmin or a fragment of plasminogenprotein sequence is used.

Applications

The method of the invention is used for prophylaxis, prevention andtreatment of periodontal disease, healing of periodontal wounds andprompting oral health for daily life. Such animals include, but are notlimited to, vertebrates such as humans and domestic animals, includingdogs, cats, horses, cows, pigs, and domesticated fowls. In oneembodiment, the methods of the invention are applied for management ofperiodontal disease in a human subject. The human or non-human subjectmay or may not suffer from a condition which impairs the healing ofperiodontal disease. In another embodiment, the methods of the inventionare applied for improving the healing of periodontal wounds. Theperiodontal wounds include, but are not limited to, traumatic wounds dueto injuries and surgical wounds. In a particular embodiment, the subjectis a human which plans to undergo, is undergoing, or has undergone,plastic surgery in the periodontal area of the mouth. In such a case, acomposition comprising, e.g., plasminogen, can be applied oradministered both prior to and/or after surgery. In a further anotherembodiment, the methods of the invention are applied for prompting oralhealth. In such a case, a composition comprising a method to increasethe level/activity of plasminogen, plasmin or miniplasmin can be appliedor administered to prompt the oral health.

Compositions and Treatments

The active agents of the invention are used for modulating thebiological activity of a drug target, and they are used in the treatmentof conditions in which degradation of ECM, host defense and/orimpairment of wound healing are observed. In particular, they may beused for preventing and treating periodontal disease, healing ofperiodontal wounds and prompting oral health.

Accordingly, the active agents of the invention may be formulated intopharmaceutical compositions for administration to subjects in abiologically compatible form suitable for administration in vivo. Bybiologically compatible form suitable for administration in vivo meant aform of the active agent to be administered in which any toxic effectsare outweighed by the therapeutic effects. The active agent may beadministered to living organisms including humans, and animals. Anactive amount of the active agent of the present invention is defined asan amount effective, at dosages and for periods of time necessary toachieve the desired result. For example, a therapeutically active amountof an active agent may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability ofantibody to elicit a desired response in the individual. Dosage rangemay be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered daily or the dose maybe proportionally reduced as indicated by the exigencies of thetherapeutic situation.

The composition(s) may be administered in a convenient manner such as byinjection (subcutaneous, intravenous, etc.), oral administration,inhalation, rectal, administration or transdermal application. Dependingon the route of administration, the active agent(s) may be coated in amaterial to protect the agent from the action of enzymes, acids andother natural conditions that may inactivate the agent. Thus, suitableroutes of administration include topical, intravenous, intramuscular,intradermal, oral, rectal, and intravaginal administration. A preferredadministration route is topical administration or oral administration.

The compositions described herein can be prepared by methods known perse for the preparation of pharmaceutically acceptable compositions whichcan be administered to subjects, such that an effective quantity of oneor more active agent(s) is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Mack Publishing Company, Easton,Pa., USA. 1985). On this basis, the compositions include, albeit notexclusively, solutions of the active agents in association with one ormore pharmaceutically acceptable vehicles or diluents, and contained inbuffered solutions with a suitable pH and iso-osmotic with thephysiological fluids.

Examples of vehicles that may be used in delivering the active agentsaccording to the invention include, but are not limited to, gel, pastes,balms, waxes, lotions, skin creams, rinsing solutions, dried powerswith/without bulking agent and various other formats for topicaladministration known in the art. The compositions may also be deliveredlocally in the form of a powder or solution sprayed, or garglingsolutions. Alternatively, the compositions of the invention may bepresent in wound dressings, pads, band-aids, gauze, or other meansapplied to the area of interest, from which they are transferred to theneeded area. Such devices also include slow-release devices, continuallyreleasing plasminogen or other agents of the invention for a prolongedperiod of time, or can include instant-release devices, which releasingplasminogen or other agents of the invention immediately at the time foruse.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of a composition of the invention, suchlabeling would include amount. frequency, and method of administration.

The compositions may be administered at regular intervals, e.g., once ortwice a day, or added in dressings or slow-release devices which arechanged as appropriate. In respect to prompt oral health, thecomposition may be administered instantly, by forming the composition atthe time of use.

The above features and many other advantages of the invention willbecome better understood by reference to the following detaileddescription when taken in conjunction with the accompanying drawings.

FIG. 1. Photographs of normal healthy gum tissue (A), inflammatorygingivitis (B) and periodontal infection periodontitis (C).

FIG. 2. Presence of plasminogen in growth medium promotes keratinocytemigration in an in vitro wound healing model.

FIG. 3. Morphology of non-treated 8-12 week old wild type andplasminogen-deficient mice jaws. Note the necrotic tissue (N, in red)and severe degradation of bone septa (B) occur in the gum tissue ofplasminogen-deficient mice, whereas the gum tissue in wild-type mice iscompletely normal. Under higher magnification (×200), the differencesbetween wild-type and plasminogen-deficient mice are even more evident(lower panels). B, bone septa. N, necrotic tissue.

FIG. 4. Morphology of non-treated 12-16 week old wild type andplasminogen deficient mice jaws. Spontaneous periodontal disease inplasminogen-deficient mice at 12-16 weeks old is more severe than thatof 8-12 weeks. Note the necrotic tissue (N, in red) and severedegradation of bone septa (B) occur in the gum tissue ofplasminogen-deficient mice, whereas the gum tissue in wild-type mice iscompletely normal. Under higher magnification (×200), the differencesbetween wild-type and plasminogen-deficient mice are even more evident(lower panels). B—bone septa, N—necrotic tissue.

FIG. 5. Morphology of non treated 16-20 week old wild type andplasminogen deficient mice jaws. Spontaneous periodontal disease inplasminogen-deficient mice at 16-20 weeks old is more severe than thatof 12-16 weeks. Note the necrotic tissue (N, in red) and severedegradation of bone septa (B) occur in the gum tissue ofplasminogen-deficient mice (upper right panel), whereas the gum tissuein wild-type mice is completely normal (upper left panel). Under highermagnification (×200), the differences between wild-typeand-plasminogen-deficient mice are even more evident (lower panels).B—bone septa, N—necrotic tissue.

FIG. 6. Bacterial recovery from saliva of wild-type,plasminogen-heterozygous and plasminogen-deficient mice.Plasminogen-deficient and plasminogen-heterozygous mice havesignificantly higher numbers of bacteria in the saliva as compared tothat of wild-type mice.

FIG. 7. Bacterial recovery from the pulled-out teeth ofplasminogen-deficient mice supplemented with human plasminogen,plasminogen-deficient mice supplemented with PBS and wild-type micewithout any treatments.

FIG. 8. Morphology of plasminogen-deficient mice supplemented with PBSor human plasminogen. Note in the PBS treated plasminogen-deficientmice, necrotic tissue was present in the gum tissue, surroundingcollagen tissue started to detach from teeth and bone resorption hadtaken place (left panels). However, supplementation of human plasminogenin plasminogen-deficient mice had completely recovered the cellular andtissue structure in the periodontal tissue. Magnification, ×50.

FIG. 9. Morphology of plasminogen-deficient mice supplemented with PBSor human plasminogen under higher magnification (×50).

FIG. 10. Morphology of plasminogen-deficient mice supplemented with PBSor human plasminogen by oral injections. Note in the PBS treatedplasminogen-deficient mice, necrotic tissue is present in the gumtissue, surrounding collagen tissue started to detach from teeth andbone resorption had taken place (upper two left panels). However,supplementation of human plasminogen in plasminogen-deficient mice hadrecovered the cellular and tissue structure in the periodontal tissue.Magnification, 100×.

FIG. 11. Morphology of non-treated 22 week old wild type (left panels)and tPA/uPA double-deficient (right panels) mice jaws. Note the necrotictissue (N, in red) and severe degradation of bone septa (B) occurs inthe gum tissue of tPA/uPA double-deficient mice, where the gum tissue inwild-type mice is completely normal. B, bone septa. N, necrotic tissue.

FIG. 12. Bacterial numbers in knee joints of plg−/− and plg+/+ mice withdifferent local and systemic treatments after inoculation of 1×10⁶ CFUof S. aureus Phillips at the knee joints.

FIG. 13. Bacterial numbers in knee joints of plg+/+ mice after localinjection with Plg (closed box) or PBS (open box) 3 days afterinoculation of S. aureus at the knee joints. Note in wild-type micelocally injected with Plg the bacterial number is significantly loweredfor 5 folds than that of wild-type locally injected with PBS.

DEFINITIONS

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terns are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them.

“A compound of the group comprising: plasminogen, plasmin, a componentof the plasminogen activation pathway, a plasminogen analogue, such asmini-plasmin, a plasmin analogue, an analogue of a component of theplasminogen activation pathway, a plasminogen activator” refers to acompound that directly or indirectly provides the effect of plasminogenor plasmin, respectively.

“A component of the plasminogen activation pathway” refers toplasminogen, Lys-plasminogen, Glu-plasminogen, variants and analogues ofplasminogen comprising one ore more domains of plasminogen such as oneore more of the kringle domains and the proteolytic domain exemplifiedby mini-plasminogen; plasmin and variants and analogues of plasmincomprising at least one ore more domains of plasmin such as one or moreof the kringle domains and the proteolytic domain, exemplified bymini-plasmin and delta-plasmin; a plasminogen activator having the finaleffect of activating plasminogen, e.g. by a cascade of events resultingin the formation or activation of plasminogen exemplified by uPA and tPAand variants and analogues of tPA and uPA comprising one ore moredomains of tPA or uPA such as one ore more of the kringle domains andthe proteolytic domain. Variants of plasminogen, plasmin, tPA and uPAinclude all naturally occurring genetic variants of human as well asother mammalian forms of these proteins, as wells as mutant variants ofthese proteins obtained by conservative amino acid replacements. An“analogue” of plasminogen or plasmin is a compound providing essentiallyan analogous effect as plasminogen or plasmin, respectively, as measuredby enzymography, ELISA (enzyme-linked immunosorbent assay) and FACS(fluorescence activated cell sorter), There is also an assay formeasuring levels of converted plasmin activity as described previously:Ny, A., Leonardsson, G., Hagglund, A. C., Hagglof, P., Ploplis, V. A.,Carmeliet, P., and Ny, T. (1999). Ovulation in plasminogen-deficientmice. Endocrinology 140, 5030-5035.). An “analogue” of a component ofthe plasminogen activation pathway is a compound providing essentiallyan analogous effect as a component of the plasminogen activation pathwayas measured by the levels of plasmin activity that this analogueactivates.

“Periodontal disease” is a common inflammatory disorder caused by theinterplay between the specific subgingival microorganisms and the hostimmune and inflammatory response. Periodontal diseases range from simplegum inflammation to serious disease that result in major damage to thesoft tissue and bone that support the teeth. In the worst cases, teethare lost. The bacteria cause inflammation of the gums that is called“gingivitis.” In gingivitis, the gums become red, swollen and can bleedeasily. Gingivitis is a mild form of gum disease that can usually bereversed with daily brushing and flossing, and regular cleaning by adentist or dental hygienist. This form of gum disease does not includeany loss of bone and tissue that hold teeth in place. When gingivitis isnot treated, it can advance to “periodontitis” (which means“inflammation around the tooth.”) In periodontitis, gums pull away fromthe teeth and form “pockets” that are infected. The body's immune systemfights the bacteria as the plaque spreads and grows below the gum line.Bacterial toxins and the body's enzymes fighting the infection actuallystart to break down the bone and connective tissue that hold teeth inplace. If not treated, the bones, gums, and connective tissue thatsupport the teeth are destroyed. The teeth may eventually become looseand have to be removed. Another type of periodontal disease,periimplantitis, occurs as a biological complication after surgicalimplantation of an alloplastic material into the jawbone.Periimplantitis is an inflammatory/infectious process affecting thetissues around an osseointegrated implant in function, resulting in lossof supporting bone. Periimplantitis may lead to complete disintegrationand implant loss even if extensive treatment aiming at resolving theperiimplant infection has been performed. Periimplantitis also happensas reversible inflammatory/infectious changes of the peri-implant softtissues without any bone loss, sometimes referred to as peri-implantmucositis. In the current patent, the definition of periodontal diseaseincludes at least periodontitis, gingivitis, periimplantitis andperi-implant mucositis.

“Infectious periodontal disease” is periodontal disease caused byinfection, in contrast to e.g. ligneous periodontitis. E.g. infectiousperiodontitis can be caused by bacterial, viral or fungal infection.

“Bacterial periodontal disease” is caused by bacterial infection.

“Periodontal wound” refers to the traumatic wounds and surgical woundsoccurring at the periodontal tissue of the mouth, including wounds atthe tissues surrounding implants in the periodontal area.

“Oral health” refers to the standard of health of the oral and relatedtissues which enables an individual to eat, speak and socialize withoutactive disease, discomfort or embarrassment and which contributes togeneral well-being. Major indications of oral health include thebacterial flora in the saliva and gum tissue, as well as the tissuenecrosis and inflammation in the gum tissue. Oral health is integral togeneral health and should not be considered in isolation.

“Derivatives of plasmin/plasminogen” refers to e.g. kringle domains ofplasmin or plasminogen, protein fragments of plasmin or plasminogen,mini-plasminogen and mini-plasmin as well as the synthetic derivativesof plasmin or plasminogen

“Mini-plasminogen” refers to the C-terminal fragment of nativeplasminogen, which includes the enzyme active site. The Mr ofminiplasminogen is 38000. Activation with urokinase or streptokinaseyields a two-chain enzyme with substrate specificity extremely similarto that of plasminogen, which is termed as ‘mini-plasmin’.

“Necrosis” refers death of tissue in the body. This happens when notenough blood is supplied to the tissue, whether from injury, radiation,or chemicals. Necrosis is not reversible. There are many causes ofnecrosis including injury, infection, cancer, infarction, invenomation,chronic wounds, ulcers and inflammation.

“Topical” and “topical application” refer to non-systemic, local,administration of an active ingredient. Thus, topical application canrefer to application of an active ingredient to the external surface ofa wound.

The “activity” of a protein or compound refers to the effect the proteinor compound has on a specific reaction, and is a measure of its abilityto affect, modulate, participate in, or promote the reaction. Generally,the activity of a protein or other compound can be measured. Forexample, in the case of enzymes such as plasmin, PA, and MMPs, andmodulators enzyme activity can be expressed as the rate at which theproduct of the reaction is produced, represented, e.g., as the amount ofproduct produced per unit of time and of enzyme (e.g., concentration orweight). In the case of modulators such as PAs, activity can refer tothe ability of the modulator to inhibit or promote, increase ordecrease, up- or down-regulate, the rate of a reaction or the amount ofproduct formed from the reaction.

A “wound” is a break in the structure of an organ or tissue, includingepithelium, connective tissue, and muscle tissue, caused by an externalagent. Examples of wounds include, but are not limited to, bruises,grazes, tears, cuts, punctures, and burns. Other particular types ofwounds are those that are a consequence of plastic surgery procedures.

“Treatment” of a subject, or “treating” a subject for a disease orcondition herein means reducing or alleviating clinical symptoms of thedisease or condition such as impaired or slow wound-healing.

“Enhancing” wound healing means increasing the speed by which the woundheals. Alternatively, “enhancing” wound healing means reducing theformations of scar tissue during or after healing.

A “subject” herein includes both human and non-human animals. Non-humananimals include, without limitation, laboratory animals such as mice,rats, rabbits, hamsters, guinea pigs, etc.; domestic animals such asdogs and cats; and farm animals such as sheep, goats, pigs, horses, andcows. A non-human animal of the present invention may be a mammalian ornon-mammalian animal; a vertebrate or an invertebrate.

A “control”, “control value” or “reference value” in an assay is a valueused to detect an alteration in, e.g., the treatment of periodontaldisease, healing of periodontal wounds and prompting oral health, or anyother assays described herein.

A subject “at risk for”, “predisposed to”, or “susceptible to” a diseaseor condition means that the risk for the individual to contract ordevelop the disease or condition is higher than in the averagepopulation.

A “deficiency” of a compound means that the amount, level, orconcentration of the compound is significantly lower than a controlvalue. For example, in a plasminogen-deficient animal, the body fluidand tissue levels of plasminogen are significantly lower than in awild-type animal.

As used herein, “about” or “approximately” shall mean within 50 percent,preferably within 20 percent, more preferably within 5 percent, of agiven value or range.

A value which is “substantially different” from another value can meanthat there is a statistically significant difference between the twovalues. Any suitable statistical method known in the art can be used toevaluate whether differences are significant or not. A “statisticallysignificant” difference means a significance is determined at aconfidence interval of at least 90%, more preferably at a 95% confidenceinterval.

Abbreviations

Abbreviations used in the present disclosure include the following:

uPA=Urokinase-type plasminogen activator;

PA=Plasminogen activator;

MMP=Matrix metalloproteinase;

TIMP=Tissue inhibitor of metalloproteinase;

tPA=Tissue-type plasminogen activator;

Plg=Plasminogen

ECM=Extracellular matrix

EXAMPLES

The invention is further described by means of the following examples.However, these examples are only illustrative of the invention, and inno way limits the scope and meaning of the invention. Indeed, manymodifications and variations of the invention will be apparent to thoseskilled in the art upon reading this specification, and can be madewithout departing from its spirit and scope.

Example 1 In Vitro Keratinocyte Migration is Dependent on the RelativeAmounts of Plasminogen

Since wound healing involves a huge number of different factors, cellsand processes, a simplified in vitro model is included to delineate thepossible plasminogen effect on cell migration.

Methods:

DOK (early neoplastic/dysplastic human oral keratinocytes) cells wereincubated in cell culture media After starving, DOK cells were incubatedin DMEM cell culture medium, containing hydrocortisone, glutamine,penicillin/streptomycin, 10% plasminogen depleted fetal bovine serum,and in the absence or presence of human plasminogen. At 0 h, a standardscratch was made on the keratinocyte layer in order to induce in vitrowound healing model. At different tine points (0 h, 12 h and 24 h) thekeratinocyte migration was documented under ZEISS microscope.

Results:

DOK (early neoplastic/dysplastic human oral keratinocytes) cellmigration seems to be almost arrested in the absence of plasminogen inthe culture media during the experimental period (FIG. 2A, upperpanels). However, in the presence of plasminogen in the culture media,keratinocyte migration appears to be enhanced as compared to that in theplasminogen-depleted media (FIG. 2A, lower-panels). After 24 hours ofplasminogen exposure (4 μM), the edges of such in vitro wounds are closeto be fused (FIG. 2B). Furthermore, cell migration rate appears to beplasminogen concentration dependent. This experiment clearly indicatesthat plasminogen is important for faster wound closure and enhancedhealing rate of damaged tissue in vitro.

Example 2 Spontaneous Development of Periodontal Disease inPlasminogen-Deficient Mice

Methods:

This experiment is dedicated to investigate the importance ofplasminogen in the development of periodontal disease by analyzingwild-type and plasminogen-deficient mice at different age.

Plasminogen-deficient (plg deficient) and wild-type (wt) mice weredivided into three age groups (5-8 mice per genotype per group): GroupI: 8-12 weeks old; Group II: 12-16 weeks old; Group III: 16-20 weeksold. The development of periodontal disease was followed by analyzingthe tissue samples of each genotype and age group.

To analyze the tissue samples, the lower and upper jaws are separatedfrom the cranium, de-fleshed from gross soft tissue such as tongue, andfixed in 4% paraformaldehyde (PFA) for 24 hours. Thereafter, sampleswere transferred to the decalcification solution to remove calcium fromthe bone tissue. After four weeks of decalcification process specimenswere embedded in paraffin and sectioned at 5 μm thickness formorphological staining. Safranin O staining was used for morphologicalanalysis, thus cartilage and mucin are stained in dark red, bonestructures and teeth are blue, and the cell nuclei are stained darkblue.

Results:

Morphological analysis of gum tissue surrounding the teeth at 8-12 weekold shows that wild type mice have no inflammation or detachment ofcollagen tissue from the tooth surface. In contrast, all the plgdeficient mice show distinct signs of initial gingivitisstages—inflammation, collagen tissue detachment from the teeth, necrotictissue formation between the teeth and degradation of bone septa andunderlying jaw bone (FIG. 3). Whereas wild-type mice have healthy oralcavity up to 20 weeks age (FIG. 3, 4, 5), in the plasminogen-deficientmice, gingivitis progresses with age to periodontitis: gum tissue isseverely inflamed, necrotic tissue forms deep in the soft tissue andevident bone septa destruction (FIG. 4, 5). These data clearlydemonstrate that plasminogen-deficient mice spontaneously develop severeperiodontal disease and the disease severity progresses with age.

Example 3 Plasminogen-Deficient Mice Have Significantly Higher Amountsof Bacteria in the Saliva Than That of Wild-type Mice

Methods:

Wild-type, plasminogen-heterozygous and plasminogen-deficient mice atage between 16-20 weeks old were used in this study (Table 1). Salivarysampling of mice was performed by collecting the saliva from the mouthwith a sterile pipette tip and transferred into anaerobic medium forimmediate culturing.

Results:

5 ul of salivary samples were successfully collected from wild-type andplasminogen-heterozygous mice. However, due to the general dryconditions of the mouths in plasminogen-deficient mice, the amount ofpossible of salivary samples resulted in variations, ranging from 1.0 ulto 5.0 ul. Bacterial recovery showed that plasminogen-deficient micehave nearly 9.0×10⁶/ml of bacteria in saliva, the highest in the threegroups and significantly higher than that of wild-type mice.Importantly, plasminogen-heterozygous mice also have significantlyhigher amounts of bacteria than that of wild-type mice (FIG. 6). Thesedata clearly indicate that plasminogen plays a critical role inmaintaining the resistance against oral bacteria. Furthermore, thenumber of bacteria seems to be critically dependent on the relativeamounts of plasminogen, which suggest a therapeutic importance forplasminogen as a novel drug to prompt oral health.

Example 4 Supplementation of Human Plasminogen in Plasminogen-DeficientMice Successfully Improves the Clinical Conditions of SpontaneousPeriodontal Disease in These Mice

Methods:

Ten plasminogen-deficient (8-12 week old) mice were randomly dividedinto plasminogen and PBS treatment groups (5 mice per group). From day 0to day 9 mice were injected intravenously daily with 100 μl of humanplasminogen (10 mg/ml) or PBS. At day 10 mice were sacrificed. For theleft side jaws, molar teeth were pulled out for the recovery ofbacteria. Right side jaws were processed for decalcification, paraffinembedding and morphological staining. Three non treated wild type miceof the same age were included as controls in the experiment.

Results:

Bacterial recovery from the pulled-out tooth samples showed thatplasminogen-deficient mice supplemented with human plasminogen havesignificantly lowered numbers of bacteria as compared to that with PBSsupplementation (FIG. 7). These data indicate that plasminogen isessential in host defense against bacterial colonization on the teeth.

As expected, severe periodontal disease occurred in all the 5 PBStreated plasminogen-deficient mice: necrotic tissue was present in thegum tissue, surrounding collagen tissue started to detach from teeth andbone resorption had taken place (FIG. 8 and FIG. 9, left panels).Plasminogen-deficient mice supplemented with human plasminogen hadcompletely recovered the cellular and tissue structure: no inflammationwas observed in the gum tissue, necrotic tissue had been removed andcollagen tissue remodeling had taken place (FIG. 8 and FIG. 9, rightpanels). For the 3 non-treated wild-type mice, the morphologicalanalysis showed similar normal tissue structure as in FIG. 3. This dataclearly show that plasminogen plays a pivotal role in maintaining normaltissue structure and function against periodontal disease.

Example 5 Local Supplementation of Human Plasminogen at the Gum Tissuein Plasminogen-Deficient Mice Successfully Improves the ClinicalConditions of Spontaneous Periodontal Disease in These Mice

Methods:

Ten plasminogen-deficient (16-20 week old) mice were randomly dividedinto plasminogen and PBS treatment groups (5 mice per group). From day 0to day 9 10 μl of human plasminogen (10 mg/ml) was locally injecteddaily to the gum tissue of both sides of the lower jaws ofplasminogen-deficient mice. For control PBS-treated group, 10 μl of PBSwas locally injected daily to the gum tissue of both sides of the lowerjaws of plasminogen-deficient mice. At day 10 mice were sacrificed andperformed for morphological studies. Three non-treated wild-type miceand three non-treated plasminogen-deficient mice were used asnon-treatment controls.

To analyze the tissue samples, the lower and upper jaws are separatedfrom the cranium, de-fleshed from gross soft tissue such as tongue, andfixed in 4% PFA for 24 hours. Thereafter, samples were transferred tothe decalcification solution to remove calcium from the bone tissue.After four weeks of decalcification process specimens were embedded inparaffin and sectioned at 5 μm thickness for morphological staining.Safranin O staining was used for morphological analysis, thus cartilageand mucin are stained in dark red, bone structures and teeth are blue,and the cell nuclei are stained dark blue.

Results:

Local injection of plasminogen at the gum tissue successfully diminishedthe severity of periodontal disease in plasminogen-deficient mice. Asexpected, severe periodontal disease occurred in all the 5 PBS treatedplasminogen-deficient mice: necrotic tissue was present in the gumtissue (N), surrounding collagen tissue started to detach from teeth andbone resorption had taken place (FIG. 10, two upper panels to the left).Plasminogen-deficient mice locally supplemented with human plasminogenhad recovered the normal cellular and tissue structure to a largeextent: low levels of inflammation were observed in the gum tissue,necrotic tissue had been removed and collagen tissue remodeling hadtaken place (FIG. 10, two upper panels to the right). For the 3non-treated wild-type and plasminogen-deficient mice, the morphologicalanalysis showed similar tissue structure as in FIG. 3, respectively.This data clearly show that local supplementation of plasminogenprovides an effective and potent way to treat periodontal disease.

Example 6 Spontaneous Development of Periodontal Disease in uPA and tPADouble-Deficient Mice

Methods:

This experiment is dedicated to investigate the importance of plasmin inthe development of, periodontal disease by analyzing the periodontaltissue in wild-type and tPA/uPA double-deficient mice. tPA and uPAdouble-deficient mice were created in our lab in order to create micelacking of plasminogen activation. These mice, although containingplasminogen in their bodies, can not convert the plasmin precursor toactive plasmin. Therefore, data from these mice can directly address ofthe importance of active plasmin in the host defense against spontaneousperiodontal disease.

The occurrence of periodontal disease in tPA/uPA double-deficient miceand their wild-type littermates at the age of 22 weeks old was followedby analyzing the tissue samples of each genotype.

To analyze the tissue samples, the lower and upper jaws are separatedfrom the cranium, de-fleshed from gross soft tissue such as tongue, andfixed in 4% paraformaldehyde (PFA) for 24 hours. Thereafter, sampleswere transferred to the decalcification solution to remove calcium fromthe bone tissue. After four weeks of decalcification process specimenswere embedded in paraffin and sectioned at 5 μm thickness formorphological staining. Safranin O staining was used for morphologicalanalysis, thus cartilage and mucin are stained in dark red, bonestructures and teeth are blue, and the cell nuclei are stained darkblue.

Results:

Morphological analysis of 22 weeks old wild-type and tPA and uPAdouble-deficient mice shows that whereas wild type mice have noinflammation or detachment of collagen tissue from the tooth (FIG. 11,left panels), all the four tPA and uPA double-deficient mice show severeperiodontal disease: gum tissue is severely inflamed, necrotic tissue(N) is formed deep in the soft tissue and evident bone septa destruction(B) had taken place (FIG. 11). These data clearly demonstrate that tPAand uPA double-deficient mice spontaneously develop severe periodontaldisease, indicating that active plasmin is also critical in themaintenance of normal periodontal health.

In the following examples, we are going to describe the findings we havein the studies of bacterial arthritis and a study we are going toperform. We believe we will get similar promising data as we had in thebacterial arthritis study. Although the data we have are from the studyof bacterial arthritis, we consider these results strongly shed light tothe promising outcoming when similar studies are performed onperiodontal area. And therefore these results suggest that localinjection of human plasminogen in the periodontal area can also restorethe normal host defense against periodontal disease in plg−/− mice. Thisconclusion is based on the consideration of following reasons:

-   -   1. The host defense mechanisms are in large extent similar in        the knee joint areas and in the periodontal areas, in that in        both situations inflammatory cells are activated and migrated to        the infectious area, bacteria are killed by the activated        inflammatory cells and other molecules, expression of cytokine        network are enhanced, necrotic tissue are removed and tissue        remodeling occurred throughout the host defense process.    -   2. S. aureus is a clinical pathogen that plays important roles        in the infectious processes during both periodontitis and        bacterial arthritis. Therefore, the data obtained from the        studies of S. aureus-induced bacterial arthritis most likely        represent a general phenomenon in spite of the specific tissue        location of the infection.    -   3. The local injection method that was used in the bacterial        arthritis model has also been used in the periodontitis study        (see Example 5) and has shown positive results where local        injection of plasminogen reduced spontaneous periodontitis in        plg−/− mice. These similarities further indicate that the        results obtained from bacterial arthritis represent a general        mechanism that also underlies the periodontal disease.

Therefore, we would like to use data from our studies on bacterialarthritis (Examples 7 and 8) to support our claims in the current patentapplication. The importance of the data from bacterial arthritis studyis that: first, the results from Example 7 indicate that local injectionof plasminogen restores the host defense capacity (e.g. killingbacteria) in a induced infection model in plg−/− mice; In addition, theresults from Example 8 indicate that local injection of plasminogen in ainduced infection model further enhances the normal host defensecapacity (e.g. killing bacteria) in normal wild-type mice. Furthermore,based on these reasons, we also include in the current patentapplication another example that we going to perform (Example 9). Inthis example, we describes a clinical model of induced periodontaldisease and we believe that application of plasminogen can restore thehost defense in plg−/− mice and further enhance the normal host defensein wild-type mice or other species.

Example 7 Local Supplementation of plg^(−/−) Mice With Human Plasminogen(hPlg) Restored the Normal Host Defense Against Bacterial Infection inthe Knee Joints

Methods

Bacterial arthritis was induced by local inoculation of 1×10⁶ CFU of S.aureus Phillips in 10 μl sterile PBS into both knee joints of mice. 15minutes after bacterial inoculation, one side of the knee joints of 6plg^(−/−) mice was supplemented with 40 μl of human plasminogen (10μg/μl in PBS, Biopool, Ume{dot over (a)}, Sweden) by local injectionsaround the knee joint tissue. Thereafter human plasminogen wassupplemented at 24-hour intervals for 7 days. As controls for localinjections, 6 plg^(−/−) mice were locally injected around the knee jointtissue with 40 ul of sterile PBS alone at 15 minutes after bacterialinoculation, and thereafter at 24-hour intervals during 7 daysexperimental period. As controls for wild-type mice, 2 plg+/+ mice weregiven 40 ul of sterile PBS alone at 15 minutes after bacterialinoculation, and thereafter every 24 hours for 7 days. As controls forplg−/− mice with systemic injections, 2 plg−/− mice were given 100 μlhuman plasminogen (10 μg/μl) intravenously 1 hour before bacterialinoculation and thereafter every 24 hours for 7 days.

Mice were sacrificed at day 7 after bacterial inoculation and the kneejoints were taken and homogenized in 1 ml sterile PBS. After serialdilutions, the solutions of homogenates were spreaded on LB agar platesand incubated at 37° C. overnight. Viable bacterial colonies were thencounted to evaluate the number of S. aureus bacteria in each homogenate.

Results

7 days of local injection of plasminogen to plg−/− mice inoculated withS. aureus successfully and significantly decreased the amounts ofbacteria to 100-folds as compared to the PBS local treatment in thesemice. Both plg−/− mice with systemic injection of human plasminogen orplg+/+ mice with local injection of PBS have also successfully killed S.aureus in their knee joints. These data (Table 1) clearly demonstratethat local injection of human plasminogen can restore the normalbacterial killing capacity in the plg−/− mice.

TABLE 1 Bacterial number in plg−/− and plg+/+ mice with different localand systemic treatments at day 3 after inoculation of 1 × 10⁶ CFU of S.aureus Phillips Mean number of Number of bacteria (Mean ± Groups samplesSD, ×10⁶ CFU) Plg−/− with local 6  0.019 ± 0.044* injection of hPlgPlg−/− with local 6 1.09 ± 0.55 injection of PBS Plg−/− with systemic 20.00075 ± 0.0011* injection of hPlg Plg+/+ with local 2  0.00065 ±0.00092* injection of PBS *P < 0.05, compared to the group of plg−/−mice with local injection of PBS.

Example 8 Local Supplementation of plg+/+ Mice With Human PlasminogenEnhances the Host Defense Against Bacterial Infection in the Knee Joints

Methods

Bacterial arthritis was induced by local inoculation of 1×10⁶ CFU of S.aureus Phillips in 10 μl sterile PBS into knee joints of mice. 15minutes after bacterial inoculation, one side of the knee joints of 7plg^(+/+) mice was supplemented with 50 μl of human plasminogen (hPlg,10 μg/μl in PBS, Biopool, Ume{dot over (a)}, Sweden) by local injectionsunder the knee skin and around the knee joint tissue. Thereafter humanplasminogen was supplemented in the same pattern at 24-hour intervalsfrom day 0 to day 2. As controls for local injections, 7 plg^(+/+) micewere locally injected under the knee skin and around the knee jointtissue with 50 ul of sterile PBS alone at 15 minutes after bacterialinoculation, and thereafter the same local injections were performed at24-hour intervals from day 0 to day 2 of the experimental period.

Mice were sacrificed at day 3 after bacterial inoculation and the kneejoints were taken and homogenized in 1 ml sterile PBS. After serialdilutions, the solutions of homogenates were spreaded on LB agar platesand incubated at 37° C. overnight. Viable bacterial colonies were thencounted to evaluate the number of S. aureus bacteria in each homogenate.

Results

Local injection at the knee joints of human plasminogen for 3 days inplg+/+ mice successfully and significantly reduced the living S. aureusnumber for 5 folds as the the control plg+/+ group treated PBS. Thesedata clearly demonstrate that human plasminogen is a potentpro-inflammatory factor that potentiate the host defense againstbacterial infection even in wild-type animal. These data (Table 2)further indicate that plasminogen is a novel anti-infectious drugcandidate for clinical use.

TABLE 2 Bacterial number in wild-type (plg+/+) mice locally injectedwith human plasminogen or PBS at day 3 after inoculation of 1 × 10⁶ CFUof S. aureus Phillips. Mean number of Number of bacteria (Mean ± Groupssamples SD, ×10⁶ CFU) Plg+/+ with local 7 0.031 ± 0.011* injection ofhPlg Plg+/+ with local 7 0.14 ± 0.047 injection of PBS Note in wild-typemice locally injected with Plg the bacterial number is significantlylowered for 5 folds than that of wild-type locally injected with PBS. *P< 0.05, as compared to the group of Plg+/+ mice with local injection ofPBS.

(Figure has same info as table, so it is not needed.)

Example 9 Supplementation of Plasminogen Restores/Enhances the HostDefense Against S. aureus-Induced Periodontal Disease in ExperimentalAnimals

Methods

The experimental model applied in this example is largely as describedbefore (31), with necessary amendment to our research settings. Since S.aureus is one of the leading pathogen in periodontal disease, we willfirst use S. aureus as the infectious bacterium in this model. However,since we believe plasminogen plays a general role in potentiating thehost defense against infection, depending on the laboratory conditions,we will probably use P. gingivalis as another infectious bacterium andperform similar studies as with S. aureus.

Thirty-six 8 week-old mice are randomly divided into three groups;ligature-infected, ligature-sham infected and controls. The mice arekept in conventional maintenance with a 12-hour light/12-hour dark cycleand are fed chow and water ad libitum.

S. aureus-Adhered Ligatures for Oral Infection

For periodontal infection S. aureus-adhered ligatures are prepared byimmersing 7 mm pieces of sterile ligature in LB broth and cultured at37° C. to late logarithmic-early stationary phase. For sham group, theligatures are processed with the above procedure but without themicroorganisms. For enumeration of the bacteria on the ligatures it issuspended in 1 ml of LB broth and vortexed for 30 seconds. Thereafterthe suspensions are diluted serially and spreaded on LB agar plates andincubated at 37° C. overnight. Viable bacterial colonies were thencounted to evaluate the number of S. aureus.

Periodontal Infection

The infection of the periodontium in the experimental- and in thesham-infected groups are performed by placing and tying the ligaturesaround molar teeth in the maxilla of the anesthetized animals. A S.aureus-adhered- or a sham- treated ligature is tied on the firstmaxillary molar (M1) in the left maxilla with the help of sterileinstruments. After the knot is tightened, the ligature is pushed intothe crevice. Control animals are not ligated nor infected with themicroorganism.

Detection and Identification of S. aureus

At the time of sacrifice, the presence of bacteria is examined by eithertaking the biofilm samples at the ligated molar area, or by pulling ofthe molar tooth carefully and in a sterile fashion. The samples areimmediately transferred into LB broth, vigorously vortexed, seriallydiluted and placed onto LB agar plates and incubated at 37° C.overnight. The total CFU on the LB agar plates are counted thereafter todetermine the number of bacteria.

Treatment with Plasminogen

After verification of the establishment of the model in mice, dailyinjection of human plg (10 ul/ul), either systemically throughintravenous injection, or locally into the marginal gingiva at the leftmaxillary M1 will commence. The starting point of the injection may beat the time of experiment starts, or during the infectious stage of theexperiment. At the end of the experiment, animals from each group willbe sacrificed for final bacteriological sampling and analysis. Themaxillary jaws separated from the cranium, de-fleshed from gross softtissue such as tongue, and fixed in 4% paraformaldehyde (PFA) for 24hours. Thereafter, samples were transferred to the decalcificationsolution to remove calcium from the bone tissue. After four weeks ofdecalcification process specimens were embedded in paraffin andsectioned at 5 μm thickness for morphological staining. Safranin Ostaining was used for morphological analysis, thus cartilage and mucinare stained in dark red, bone structures and teeth are blue, and thecell nuclei are stained dark blue.

Results

Based on our previous experience from plasminogen treatment of plg−/−mice, where injections both systemically and locally has reconstitutedthe gingival inflammation, the normal host defense (e.g. killing ofbacteria) and proper periodontal re-attachment in these animals whichhave spontaneous periodontal disease, we strongly predict a similarresponse to the plasminogen treatment of plg−/− mice in the inducedperiodontal disease model as described above. Furthermore, based on thedata of our previous study on another infection model, bacterialarthritis model, where local injection of plasminogen enhances thenormal host defense against infectious bacteria in plg+/+ mice, westrongly predict that local treatment of plasminogen in plg+/+ miceduring the induced periodontal disease model described above will alsoenhance the normal host defense in plg+/+ mice. And thus, all these datawill show a strong indication that plasminogen is a novel drug candidateto preventing and treating periodontal disease, improving healing ofperiodontal wounds and promoting oral health

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1-42. (canceled)
 43. A method of prophylaxis, prevention and/ortreatment of infectious periodontal disease comprising administering apharmaceutical composition comprising a compound selected from the groupconsisting of plasminogen, Lys-plasminogen, Glu-plasminogen, variants ofplasminogen comprising one or more of the kringle domains and theproteolytic domain, mini-plasminogen, and kringle domains of plasminogenin an effective amount to a subject in need of thereof.
 44. Method ofclaim 43, wherein the periodontal disease is, or is caused by, abacterial infection.
 45. Method of claim 43, wherein the periodontaldisease is selected from the group consisting of periodontitis,gingivitis, necrotizing gingivitis, periimplantitis, and peri-implantmucositis.
 46. Method of claim 43, wherein the subject is a human, andthe compound is human plasminogen.
 47. Method of claim 43, wherein thesubject is a non-human mammal.
 48. Method of claim 43, wherein thepharmaceutical composition further comprises a pharmaceuticallyacceptable carrier.
 49. Method of claim 43, wherein the pharmaceuticalcomposition is selected from the group consisting of an aqueoussolution, a gargling solution, a gel, a lotion, a balm, a powder, apaste, a toothpaste, a bandage, and a wound dressing.
 50. Method ofclaim 43, wherein the pharmaceutical composition is administered by amethod selected from the group consisting of spray, topical, oral,local, and systemic administration.
 51. Method of claim 50, wherein thepharmaceutical composition for topical administration comprises fromabout 1 μg to about 500 mg plasminogen per square centimeter of theapplication area.
 52. Method of claim 43, wherein the administering isrepeated at least once.
 53. Method of claim 52, wherein theadministering is repeated daily.
 54. Method of claim 43, furthercomprising a step selected from the group consisting of activatinginflammatory cells, enhancing keratinocyte migration, reducing bacterialgrowth, removing necrotic tissue, improving tissue remodeling, andenhancing cytokine expression.
 55. A pharmaceutical composition for thetreatment, prophylaxis and/or prevention of infectious periodontaldisease caused by bacterial, viral or fungal infection comprising aneffective amount of a compound selected from the group consisting ofplasminogen, Lys-plasminogen, Glu-plasminogen, variants of plasminogencomprising one or more of the kringle domains and the proteolyticdomain, mini-plasminogen, and kringle domains of plasminogen.
 56. Thepharmaceutical composition according to claim 55, wherein the compoundis human plasminogen.
 57. A method of promoting the healing ofinfectious periodontal wounds comprising administering a pharmaceuticalcomposition comprising a compound selected from the group consisting ofplasminogen, Lys-plasminogen, Glu-plasminogen, variants of plasminogencomprising one or more of the kringle domains and the proteolyticdomain, mini-plasminogen, and kringle domains of plasminogen in aneffective amount to a subject in need thereof.
 58. Method of claim 57,wherein the infectious periodontal wound is a wound caused by injury ora wound caused by periodontal surgery or plastic surgery.
 59. Method ofclaim 57, wherein the subject is a human, and the compound is humanplasminogen.
 60. Method of claim 57, wherein the subject is a non-humanmammal.
 61. Method of claim 57, wherein the pharmaceutical compositionfurther comprises a pharmaceutically acceptable carrier.
 62. Method ofclaim 57, wherein the pharmaceutical composition is selected from thegroup consisting of an aqueous solution, a gargling solution, a gel, alotion, a balm, a powder, a paste, a toothpaste, a bandage, and a wounddressing.
 63. Method of claim 57, wherein the pharmaceutical compositionis administered by a method selected from the group consisting of spray,topical, oral, local, and systemic administration.
 64. Method of claim63, wherein the pharmaceutical composition for topical administrationcomprises from about 1 μg to about 500 mg plasminogen per squarecentimeter of the application area.
 65. Method of claim 57, wherein theadministering is repeated at least once.
 66. Method of claim 65, whereinthe administering is repeated daily.
 67. Method of claim 57, furthercomprising a step selected from the group consisting of reducing fibrindeposition, promoting keratinocyte migration, enhancing cytokineexpressions, removing necrotic tissue, activating inflammatory cells,and improving tissue remodeling.
 68. A pharmaceutical composition forpromoting the healing of infectious periodontal wounds, which comprisesan effective amount of a compound selected from the group consisting ofplasminogen, Lys-plasminogen, Glu-plasminogen, variants of plasminogencomprising one or more of the kringle domains and the proteolyticdomain, mini-plasminogen, and kringle domains of plasminogen.
 69. Thepharmaceutical composition according to claim 68, wherein the compoundis human plasminogen.